Non-Hodgkin lymphoma

Context.-Molecular genetic analyses have been predicted to improve the diagnostic accuracy of fine-needle aspiration of B-cell non-Hodgkin lymphoma.

Objective.-To determine the value of routine molecular genetic assays, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), in the diagnosis of Bcell non-Hodgkin lymphoma by fine-needle aspiration (FNA).

Design.-A multiparametric method, including cytology, flow cytometry, PCR, and FISH, was prospectively evaluated in the diagnosis of B-cell non-Hodgkin lymphoma by FNA. Aspirates from 30 consecutive patients with suspected hematolymphoid malignancies were collected. All aspirates were triaged through a uniform program including cell-size analysis, B- and T-cell clonality studies, flow cytometric immunophenotyping, and bcl-1 and bcl-2 gene rearrangements by PCR and FISH. After completion of FNA evaluations, FNA results were compared with diagnoses from prior or subsequent surgical biopsies.

Results.-Monoclonal B-cell populations were detected in 18 of 20 B-cell non-Hodgkin lymphomas by flow cytometry and PCR. bcl-1 gene rearrangement was detected in 2 of 2 cases of mantle cell lymphoma. bcl-2 rearrangement was detected in 5 cases including 4 of 4 low-grade follicular lymphomas and 1 transformed follicular lymphoma. By incorporating the results of molecular genetic and ancillary diagnostics, a definitive classification was reached in 12 cases of B-cell non-Hodgkin lymphoma by FNA, including all cases of low-grade follicular lymphoma (4/4) and mantle cell lymphoma (2/2) and approximately 50% of small lymphocytic lymphoma (2/4) and large B-cell lymphoma (4/8). Ten of the 12 cases with a final classification reached by FNA had either prior or follow-up surgical biopsies, and all 10 cases showed agreement between the diagnoses rendered on FNA and surgical biopsies.

Conclusions.-With proper handling and management of specimens, FNA can routinely provide samples adequate for molecular genetic studies, in addition to cytomorphology and flow cytometry, making it possible to consistently render accurate and definitive diagnoses in a subset of Bcell non-Hodgkin lymphomas. By incorporating FISH and PCR methods, FNA may assume an expanded role for the primary diagnosis of B-cell non-Hodgkin lymphoma.

(Arch Pathol Lab Med. 2004;128:1395-1403)

Fine-needle aspiration (FNA) is increasingly used in the evaluation of both new lymphadenopathy and recurrent lymphomas. Although the role of FNA in the assessment of recurrent lesions has been well established, the value of this technique in the primary diagnosis of B-cell non-Hodgkin lymphoma (B-NHL) continues to be a subject of debate. For patients presenting with new lymphadenopathy, FNA is commonly used as an initial guide to medical management or further surgical biopsy. However, broader implementation of FNA in the classification of BNHL has remained constrained by the considerable cytologie overlap and immunophenotypic heterogeneity among B-NHL subtypes.

These factors have been particularly limiting in the analysis of small B-NHL. Given the extensive morphologic overlap between reactive processes and small B-cell lymphomas, and among lymphoma subtypes, cytomorphologic evaluation alone provides a very limited basis for the diagnostic classification of small B-cell lymphomas.1 For example, in the case of mantle cell lymphoma (MCL), the spectrum of cytologie variants includes small cell, centrocytic cell, and blastoid forms that mimic small lymphocytic lymphoma (SLL), follicular lymphoma (FL), and lymphoblastic lymphoma, respectively.21 Given the cytologie heterogeneity, immunophenotyping using flow cytometry or immunohistochemistry has become standard practice, both for the determination of monoclonality and diagnostic subclassification. However, the presence of immunophenotypic variability among small B-NHL subtypes is an additional confounding variable. For example, although the expression of CDS is typically used as a diagnostic criterion for MCL and SLL, both types may lack CDS expression4,5 and other B-NHLs may occasionally express CDS.6,7 Mantle cell lymphoma and SLL are phenotypically differentiated by differences in CD23 expression, but CD23 may be weakly expressed by both types.8,9 As a third example, although FL is usually characterized by CD10 positivity, the intensity of CD10 expression by FL has been shown to be variable, and partial or negative CD10 expression is not uncommon.10,11

Under current (2001) World Health Organization criteria, molecular analysis has assumed a greater role in BNHL diagnosis. The most consistent chromosomal abnormalities in B-NHL, reciprocal translocations t(11;14)(q13; q32) and t(14;18)(q32;q21) in MCL and FL, respectively, are now viewed as highly specific markers, present in greater than 90% of the cases and readily detected with fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Both techniques have become widely used as ancillary studies for surgical biopsy specimens. Previous reports have shown that FISH can be performed on FNA specimens.12 However, there is only limited published data regarding the diagnostic utility of FISH-based detection of these translocations.13,14 Additionally, few prior FNA studies have examined PCR assays of these genotypic markers.15,16

The aim of this prospective study was to determine whether routine molecular genetic evaluation would allow FNA to achieve the diagnostic accuracy of surgical biopsy in a subset of B-NHL cases. We predicted that the incorporation of molecular tests, as part of a multiparametric approach, would improve the utility of FNA in the primary evaluation of patients having clinically suspected hematolymphoid malignancies.

MATERIALS AND METHODS

Patient Samples

From December 2001 to April 2003, patients who underwent FNA evaluation for suspected hematolymphoid malignancies at Duke University Medical Center were requested to enroll in the study. Written informed consent was obtained from all 30 study participants. Approval by Duke University Medical Center Institutional Review Board was obtained before initiation of the study. Standard FNA procedures were performed as previously described by Liu et al17 and included both percutaneous and imageguided procedures (ultrasound or computed tomography). Several cell smears were made from the initial pass. The smears were stained with Papanicolaou and Diff-Quick (rapid Romanowsky) stains and reviewed on-site by a cytopathologist for assessment of sample adequacy and for rapid cytologic interpretation. If hematolymphoid malignancy was suspected, depending on overall cellularity and location of the lesion, 1 to 3 additional passes were made for extra smears and cell suspensions. Two unstained smears were air dried and stored at room temperature for FISH studies. Cell suspensions were prepared in 15 to 20 mL normal saline and were equally divided into 2 aliquots for flow cytometry and PCR analyses.

Cytologic Examination

Papanicolaou and Diff-Quick stains were examined for specimen adequacy, cell size, morphology, and extracellular features. Cell-size analysis was performed according to the methods described previously.18 A large cell was defined as being equal to or greater than twice the diameter of a small lymphocyte, with open chromatin and 1 to several nucleoli. An intermediate cell was defined as being between 1.5 and 2 times the diameter of a small lymphocyte (Figure 1, A and B).

Flow Cytometry

Three-color flow cytometric analysis was performed on a single-cell aliquot using direct immunofluorescence staining with antibodies preconjugated with fluorescein isothiocyanate, phycoerythrin, or peridinin chlorophyll protein, according to previously described methods.18 Monoclonal antibodies and their combinations included CD19/κ/λ, CD19/CD5/CD3, CD19/CD10/ CD33, CD19/CD20/CD23, CD45/CD38/CD56, CD3/CD4/ CDS, and CD3/CD2/CD5. Antibody panels were selected on a case-by-case basis during the diagnostic investigation of patient samples based on clinical and laboratory data, past history of lymphoma, and cytomorphologic features. Data were collected on a FACScaliber analyzer (Becton Dickinson, San Diego, Calif) and analyzed with CellQuest software (Becton Dickinson). Using flow cytometry, we evaluated 3 parameters. The first was cell size as a percentage of large, small, and intermediate cells, performed according to the previously published method.18 Briefly, the percentages of large cells were determined by comparison with the reactive T-cell population and were calculated by CellQuest software statistics. A small-cell population was defined as complete overlap of the neoplastic population with the reactive T cells on forward scatter histogram. A large-cell population was defined as a complete shift of the neoplastic population from the reactive T-cell population without overlap (Figure 2). The second and third parameters were clonality by monotypic immunoglobulin light chain, κ or λ, or κ/λ double-negative events and antigen expression, respectively.

Polymerase Chain Reaction

Cells for molecular studies were prepared by centrifugation and pellet retrieval, followed by either dilution in cell lysis solution or storage at -80°C. Genomic DNA was prepared from each sample using the Puregene DNA purification kit according to the manufacturer's protocols (Centra Systems, Minneapolis, Minn). After extraction, DNA was rehydrated, then quantified by ultraviolet spectroscopy, following standard laboratory methods.

Immunoglobulin heavy chain (IgH) and T-cell receptor γ gene (TCR-γ) rearrangements were evaluated by DNA PCR according to the manufacturer's instructions (InVivoScribe Technologies, Carlsbad, Calif). Briefly, the Immunoglobulin Gene Rearrangement Assay kit framework regions 1 (FRI) and 3 (FRIII) standardized master mixes were used for PCR analysis of B-cell clonality (InVivoScribe). TCRγ assay kit master mix 1 and master mix 2 were used to amplify patient DNA and assess the presence of clonal T-cell populations (InVivoScribe). Polymerase chain reaction products were analyzed on an ABI 310 gene analyzer (Applied Biosystems, Foster City, Calif). Clonal B-cell and T-cell populations were determined by the detection of 1 or 2 peaks within the defined regions. Results for B- and T-cell assays were reported as clonal (1-2 clear peaks), oligoclonal (3 or more clear peaks), polyclonal, or indeterminate for clonality according to standard laboratory protocols (Duke University Health Systems Molecular Diagnostics Laboratories, Durham, NC). Appropriate positive and negative controls were used (Figure 3, A).

Polymerase chain reaction assays for the detection of bcl-l and bcl-2 gene rearrangements were performed according to previously described methods.19-21 Briefly, primer pair MCL-1/J^sub H^ was used for PCR screening for bcl-1 rearrangement t(11;14)(q13;q32) at the major translocation cluster.19 bcl-2 rearrangement t(14; 18)(q32;q21) was detected using primers to the major breakpoint region, MBR/J^sub H^, and the minor translocation cluster, MCS/J^sub H^. After electrophoretic separation on a 1.5% agarose gel, PCR products were transferred to a nylon membrane. Products of bcl-1 and bcl-2 amplification were hybridized with ^sup 32^KP-labeled oligoprobes specific for bcl-l MTC (P3 5'-AGGCTGCTGTACACATCG-3'), bcl-2 MBR (MBI),20 and bcl-2 MCS (MCI).20 The predicted bcl-l MTC amplicon was approximately 500 bp.19 MBR and MCS products were predicted to occur between 80 bp and 2 Kb, based on previously published data.20 Genomic DNA extracted from bcl-1 and bcl-2 positive cell lines and diluted 1:10 with normal DNA were used as positive controls. Amplifiable patient DNA was determined using primers to the human β-globin gene (Figure 3, B).

FISH Studies

Slides for FISH analysis of chromosomal rearrangements t(14; 18)(q32;q21) and t(11;14)(q13;q32) were prepared from unstained air-dried FNA smears. The slides were fixed in 3:1 methanol-acetic acid followed by a 20-minute digestion in 10% pepsin/ 0.01N HCl. The probes were then applied and denatured at 75°C for 1 minute and then incubated at 37°C for 16 to 18 hours. Probes used in this study were dual color, dual fusion IgH/bcl-2 and IgH/CCND1 (Vysis, Inc, Downers Grove, Ill). The slides were counterstained with 4,6-diamidino-2-phenylindole in antifade solution (DAPI). Interphase nuclei were analyzed using a Zeiss Axiophot microscope equipped with a Chroma 83000 filter set and Quips Pathvysion Software (Applied Imaging, Santa Clara, Calif). Before counting, the hybridization signals were screened for the presence of variant signal patterns. In each case, 100 to 200 nuclei were scored. The normal range for each probe was determined using slides from patients with no hematologic malignancies. Appropriate positive controls were selected from archival material of patients with prior cytogenetic evidence of the reciprocal translocations. Representative images were captured using a Sensys charge-coupled device camera (Roper Scientific Inc, Tucson, Ariz) (Figure 1, C and D).

RESULTS

Aspirates from 30 adult patients undergoing diagnostic FNA for the evaluation of clinically suspected hematolymphoid malignancies were collected. The FNA specimens were from nodal and extranodal sites from 14 male and 16 female patients, ages 25 to 84 (mean age, 62.4). Of these 30 biopsies, 14 were performed by radiologists using either computerized tomography (11) or ultrasound (3). Specifically, of the 14 image-guided procedures, 12 involved sampling of deep-seated sites including the chest (1), abdomen (10), and pelvis (1). The remaining 16 specimens were collected from superficial sites overlying the neck (7), axilla (4), and inguinal regions (5) by trained cytopathologists. Twenty-seven cases had either prior or follow-up surgical biopsies. However, all FNA diagnoses were rendered independently of prior diagnoses or followup information. Only after FNA evaluation had been completed were FNA results then compared with the results from any surgical biopsies.

Cytologic examination and flow cytometry were performed for all the cases. Polymerase chain reaction for Band T-cell clonality, FISH and PCR for bcl-1 and bcl-2 rearrangements were performed on every case when sufficient material was available. Final classification by FNA was reached in 12 cases of B-NHL (Table 1, cases 1-12) including 4 of 4 low-grade FL, 2 of 2 MCL, 2 of 4 SLL, 3 of 7 diffuse large B-cell lymphoma (DLBL), and 1 DLBL/ follicular large cell lymphoma. All the low-grade FL and MCL showed a predominance of small cells (less than 20% large cells) by cytology and flow cytometry, monoclonal B cells by flow cytometry or PCR, and bcl-2 and bcl-l rearrangements, respectively, by FISH or PCR. The MCLs, in addition, showed CDS positive and CD23 dimly positive or negative phenotype. Two SLLs showed less than 20% large cells, monoclonality, CDS positive and CD23 positive phenotype, and lacked both bcl-2 and bcl-l rearrangements. Of the 4 large B-cell lymphomas diagnosed by FNA, 3 were DLBL. Multi-parametric analysis showed greater than 40% large cells by cytology and flow cytometry, monoclonality by flow cytometry or PCR, and no bcl2 or bcl-1 rearrangements. The fourth large B-cell lymphoma demonstrated bcl-2 rearrangement by FISH. Because bcl-2 rearrangement may be found in both follicular large cell lymphoma and DLBL, a diagnosis of DLBL/FL III (follicular lymphoma grade III) was made in this case (Table 2).

In the 12 cases of B-NHL with a final classification reached by FNA, 10 cases had either prior or follow-up surgical biopsies. In each of these 10 cases, there was complete agreement between diagnoses rendered by FNA and surgical biopsies (Table 1).

An additional 6 cases were diagnosed as B-NHL, with evidence of monoclonality, but further classification could not be made by FNA (Table 1, cases 13-18). Included among these 6 cases were 2 cases of SLL with CD5 expression but dim or undetermined CD23 expression (cases 13 and 14), 2 DLBLs in which the number of large cells estimated by flow cytometry did not fulfill the criteria for large cell lymphoma ( cases 17 and 18), and 2 cases of unclassifiable small B-NHL with predominance of small cells by cytology and flow cytometry, but negative CDS and CDlO expression ( cases 15 and 16). Neither bcl-2 or bcl-l rearrangement nor T-cell clonality was detected in any of these 6 cases.

The remaining 12 cases not found to be diagnostic of BNHL by FNA included reactive lymphoid hyperplasia, BNHL, T-cell lymphoma, Hodgkin lymphoma, and other hematopoietic malignancies (Table 1, cases 19-30). Clonal B-cell populations were not detected in these cases by either flow cytometry or PCR, including 2 NHL cases ( cases 23 and 24). Clonal T-cell populations were detected only in the 2 cases of T-cell lymphoma. bcl-2 and bcl-l rearrangements were not detected in any of these 12 cases. In 2 lymph node biopsies from patients with prior diagnoses of carcinoma ( cases 19 and 20), flow cytometry and PCR showed a polyclonal B-cell pattern. However, metastatic carcinoma was not identified in these cytologic smears. cases 21 through 24 were suspected NHLs by FNA with a predominance of lymphocytes by cytology, but indeterminate for clonality owing to insufficient B cells ( case 21) or a predominance of nonviable cells (cases 22-24).

COMMENT

Diffuse large B-cell lymphoma and FL rank as the first and second most frequent subtypes of NHL in Western countries, each comprising approximately 30% of all NHL cases. Although MCL is less common, it accounts for a clinically important subset, significant for its poorer prognosis. A fourth subset, small lymphocytic lymphoma/ chronic lymphocytic leukemia, typically presents in the leukemic phase. However, diagnosis may be more complicated when it presents as isolated lymphadenopathy. Given the variability in prognoses and differing therapeutic objectives for B-NHL subtypes, accurate tissue diagnosis is the cornerstone of medical treatment.22

Traditionally, there has been considerable reservation regarding the use of FNA for the primary diagnosis of BNHL. This view has persisted among many pathologists and clinicians because of the morphologic and phenotypic heterogeneity of these disease processes. In the case of large B-cell lymphoma, criteria for FNA diagnosis have previously been more extensively investigated and include transformed lymphocyte count,23 flow cytometric cell size analysis,18 DNA ploidy,24 and proliferation markers.25,26 However, studies evaluating the diagnosis and subclassification of small B-cell lymphomas by FNA have thus far remained limited.27-30 Gong et al13 recently compared the utility of FISH and flow cytometry in a series of FL and DLBL evaluated by FNA. They found that, in FL, detection of t(14;18) by FISH was a slightly more sensitive (85%) diagnostic marker than demonstration of CD10 by flow cytometry (75%). Bentz et al14 attempted the application of FISH on FNA specimens in MCL and demonstrated t(11;14) in all 8 FNA and 2 body-cavity fluid cases studied. Few reports have attempted to incorporate PCR in the diagnosis of FL and MCL by FNA. Alkan et al15 evaluated 4 cases of FL on archival air-dried FNA samples, bcl-2 rearrangement was detected in 2 cases by PCR. Hughes et al16 attempted PCR and chromosome karyotyping in a blastic MCL by FNA, although bcl-1 rearrangement was not detected in that case.

In this study, we prospectively evaluated 30 suspected cases of hematolymphoid malignancy by FNA. Both PCR and FISH methods were used in the detection of t(14; 18)(q32;q21) and t(11;14)(q13;q32) when materials were available. In the 4 cases of FL, only 1 case was bcl-2 positive by both FISH and PCR, while for the other 3 cases, bcl-2 rearrangement was detected by either PCR (2 cases) or FISH (1 case). In the 2 MCL cases in this shady, bcl-l rearrangement was detected by FISH but not by PCR (PCR performed in 1 case).

Differences in FISH and PCR detection rates may be explained by the specific limitations of each method. Interphase FISH, which uses gene-spanning probes, has the potential to detect bcl-2 rearrangement in nearly all cases of FL that harbor this translocation.31,32 However, the sensitivity of FISH is significantly decreased when smears contain low numbers of tumor cells admixed with nontumor tissue. In contrast, PCR may potentially achieve greater sensitivity than FISH, given a limited tumor sample. Using primer sets to MBR and MCS regions, PCR has been shown to detect bcl-2 rearrangement in as low as 0.1% to 0.01% tumor cells.20,33 However, because of variations in translocation breakpoints and mutations involving primer binding sequences, PCR has been shown to detect only about 50% of bcl-2 rearrangement in FL.20,34

Similar sensitivities of FISH and PCR methods have previously been described for the detection of bcl-l rearrangements in MCL.35,36 The PCR method used in this study showed a 40% sensitivity in the detection of t(ll; 14) in a series of surgically removed and histologically confirmed MCL cases in our laboratory (unpublished data), which is consistent with previously published results.35,37,38 The relatively low sensitivity of this method is primarily due to the wider variation of translocation breakpoints on chromosome 11. Although PCR failed to detect bcl-l rearrangement in one of our studied MCL cases, we speculate, based on our bcl-2 FL results, that a larger case series would show that PCR and interphase FISH are not redundant, but complementary techniques. Incorporation of both methods may increase the overall sensitivity of detection for aspirated samples.

It is essential that the demonstration of these rearrangements, in particular bcl-2, be interpreted in the context of a monoclonal B-cell population. Previous studies have demonstrated that the bcl-2 rearrangement can be detected in reactive lymphoid processes,39,40 whereas both bcl-2 and bcl-l rearrangements have been found in association with other lymphoma subtypes such as DLBL and multiple myeloma, respectively.41,42 However, a careful morphologic and phenotypic examination will often exclude these lesions. Detection of bcl-2 rearrangement in an aspirate of large B cells may also complicate FNA diagnosis. Both high-grade FL and DLBL may harbor this translocation. Distinguishing between these 2 entities currently requires a surgical biopsy. We encountered this problem with case 12 where a final diagnosis of diffuse large B-cell lymphoma/follicular lymphoma, grade III, was rendered. In this case, we anticipate that a surgical biopsy will be necessary for definitive classification and to guide further therapeutic intervention.

Previously, a variety of methods have been used to determine monoclonality from FNA specimens, including Southern blot,43-45 PCR,30,46-49 in situ hybridization,50 flow cytometry,51 and immunocytochemistry.13 In this study, we evaluated 2 methods, flow cytometry and PCR, and compared their relative utility for the assessment of B-cell clonality. In the 12 cases of B-NHL with a final diagnosis made by FNA, a monoclonal κ or λ population was detected by flow cytometry (including 1 case with negative κ and λ expression; Table 2), and clonal populations were detected in 7 of 10 cases by PCR. It is common knowledge that PCR may not always detect immunoglobulin gene rearrangement, primarily because of the diversity of immunoglobulin gene sequences. The 70% detection rate in our study is consistent with the sensitivities previously reported for surgical biopsies using similar methods.52,53 Davidson et al54 drew similar conclusions and suggested that the combination of flow cytometry and PCR might increase the overall sensitivity of clonal detection. However, because flow cytometry is highly sensitive and widely available, we recommend that the routine detection of monoclonal B cells in aspirates should first be attempted using flow cytometry. Polymerase chain reaction should be reserved for cases in which flow cytometry fails to detect a monoclonal population. Nethertheless, demonstration of monoclonality by either flow cytometry or PCR is an essential step, which should be performed before further immunophenotypic or genotypic studies.

Although the focus of this study was primarily on the differential diagnosis of small B-NHLs, 7 diffuse large B-cell lymphomas and 1 transformed large B-cell lymphoma were included in this study. A definitive diagnosis by FNA was reached in 50% of large B-cell lymphomas, compatible with the conclusion from a previous study by us.18 Lack of a sufficient number of large cells and lack of a detectable clonal population were so far the most common causes of failed FNA diagnosis for DLBL.18,55,56 Of the 4 cases of nondiagnostic DLBL in this study, 3 cases showed monoclonal populations by flow cytometry, but the number of large cells did not fulfill the criteria of DLBL (Table 1, cases 15, 17, and 18) including case 15 in which a predominance of small cells was detected. The fourth case failed to demonstrate clonality by flow cytometry and PCR owing to a lack of viable cells and lack of amplification, respectively (Table 1, case 24). It is interesting that case 15 showed a predominance of small cells by FNA on a cervical lymph node, but a follow-up surgical biopsy of an inguinal lymph node revealed a diffuse large B-cell lymphoma. It is not uncommon to see NHL with different histologie morphology in different sites. In this case, the DLBL might likely be a transformed lymphoma that originated from a small cell-type NHL.

In this study we propose a combined diagnostic algorithm including cytology, flow cytometry, and molecular evaluation for the diagnosis of B-NHL by FNA (Figure 4). Central to this algorithm is the disciplined handling of FNA specimens. It is our recommendation that, in every case of suspected lymphoma where sufficient material is available, cells should be aliquoted for PCR and FISH, in addition to flow cytometry and cytologie evaluation. Although molecular genetic studies will certainly not be necessary in every case, care must be taken to initially secure and appropriately process material from all aspirates so that it is available should the need arise. Furthermore, despite the relatively limited number of lymphomas, this study clearly demonstrated that careful and deliberate handling of FNA specimens provides sufficient material for a multilayered diagnostic analysis.

Following cytologic identification of a lymphoid population, the initial step in our algorithm is the determination of cell size. Cell-size analysis should be performed by evaluation of transformed lymphocyte count in cytologic smears and by flow-cytometry cell-size analysis. Analysis of cell-size categorizes NHL as large cell (>40% large cells), small cell (

This multi-parametric, FNA-based algorithm categorized the majority of small B-cell lymphomas, including all low-grade FL, MCL, and about half of SLL. This study suggests that FNA could become a first-line approach to the diagnosis and classification of common types of B-NHL, obviating the risk and expense of surgical biopsies for a significant subset of patients. Larger scale investigations may be necessary to better understand the true costs, benefits, and limitations of this algorithm. Other small B-cell subtypes, including marginal zone lymphoma and lymphoplasmacytic lymphoma, currently lack specific surface markers and consistent genetic markers, and continue to require surgical biopsy for definitive classification. Until new specific surface and genetic markers are identified, the value of FNA in the classification of these specific lymphomas will remain limited.

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Anne M. Safley, MD; Patrick J. Buckley, MD, PhD; Andrew J. Oeager, MD; Rajesh C. Dash, MD; Leslie G. Dodd, MD; Barbara K. Goodman, PhD; Claudia K. Jones, MD; Anand S. Lagoo, MD, PhD; Timothy T. Stenzel, MD, PhD; Weihua Wang, MD; Bill Xie, MD, PhD; Jerald Z. Gong, MD

Accepted for publication July 30, 2004.

From the Department of Pathology, Duke University Medical Center, Durham, NC. Dr Stenzel is now with Vysis, Incorporated, Downers Grove, III.

All conclusions and interpretations in this publication with respect to the College of American Pathologists' database are those of the authors and not those of the College.

Dr Stenzel has declared that, following completion of this work, he began working for Vysis, provider of the FISH probe purchased for this study. He currently serves as medical director at Vysis. All other authors have no relevant financial interest in the products or companies described in this article.

Reprints: Jerald Z. Gong, MD, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710 (e-mail: gong0001@mc.duke.edu).

Copyright College of American Pathologists Dec 2004
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